Gene gun

In genetic engineering, a gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for delivering exogenous DNA (transgenes) to cells. The payload is an elemental particle of a heavy metal coated with DNA (typically plasmid DNA). This technique is often simply referred to as biolistics. In genetic engineering, a gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for delivering exogenous DNA (transgenes) to cells. The payload is an elemental particle of a heavy metal coated with DNA (typically plasmid DNA). This technique is often simply referred to as biolistics. This device is able to transform almost any type of cell, including plants, and is not limited to transformation of the nucleus; it can also transform organelles, including plastids. The gene gun was originally a Crosman air pistol modified to fire dense tungsten particles. It was invented by John C Sanford, Ed Wolf and Nelson Allen at Cornell University, and Ted Klein of DuPont, between 1983 and 1986. The original target was onions (chosen for their large cell size) and it was used to deliver particles coated with a marker gene. Genetic transformation was then proven when the onion tissue expressed the gene. The earliest custom manufactured gene guns (fabricated by Nelson Allen) used a 22 caliber nail gun cartridge to propel an extruded polyethylene cylinder (bullet) down a 22 cal. Douglas barrel. A droplet of the tungsten powder and genetic material was placed on the bullet and shot down the barrel at a lexan 'stopping' disk with a Petri dish below. The bullet welded to the disk and the genetic material blasted into the sample in the dish with a doughnut effect (devastation in the middle, a ring of good transformation and little around the edge). The gun was connected to a vacuum pump and was under vacuum while firing. The early design was put into limited production by a Rumsey-Loomis (a local machine shop then at Mecklenburg Rd in Ithaca, NY, USA). Later the design was refined by removing the 'surge tank' and changing to nonexplosive propellants. DuPont added a plastic extrusion to the exterior to visually improve the machine for mass production to the scientific community. Biorad contracted with Dupont to manufacture and distribute the device. Improvements include the use of helium propellant and a multi-disk-collision delivery mechanism. Other heavy metals such as gold and silver are also used. Gold may be favored because it has better uniformity than tungsten and tungsten can be toxic to cells, but its use may be limited due to availability and cost. A construct is a piece of DNA inserted into the target's genome, including parts that are intended to be removed later. All biolistic transformations require a construct to proceed and while there is great variation among biolistic constructs, they can be broadly sorted into two categories: those which are designed to transform eukaryotic nuclei, and those designed to transform prokaryotic-type genomes such as mitochondria, plasmids or plastids. Those meant to transform prokaryotic genomes generally have the gene or genes of interest, at least one promoter and terminator sequence, and a reporter gene; which is a gene used to enable detection or removal of those cells which didn't integrate the construct into their DNA. These genes may each have their own promoter and terminator, or be grouped to produce multiple gene products from one transcript, in which case binding sites for translational machinery should be placed between each to ensure maximum translational efficiency. In any case the entire construct is flanked by regions called border sequences which are similar in sequence to locations within the genome, this allows the construct to target itself to a specific point in the existing genome. Constructs meant for integration into a eukaryotic nucleus follow a similar pattern except that: the construct contains no border sequences because the sequence rearrangement that prokaryotic constructs rely on rarely occurs in eukaryotes; and each gene contained within the construct must be expressed by its own copy of a promoter and terminator sequence. Though the above designs are generally followed, there are exceptions. For example, the construct might include a Cre-Lox system to selectively remove inserted genes; or a prokaryotic construct may insert itself downstream of a promoter, allowing the inserted genes to be governed by a promoter already in place and eliminating the need for one to be included in the construct.